Transfer of the plasmid for exfoliative toxin B synthesis in mixed cultures on nitrocellulose membranes.

Plasmid pRW002 carries genetic determinants for exfoliative toxin B and bacteriocin R1 synthesis. When a donor strain carrying plasmid pRW002 was mixed with a plasmidless recipient strain on a nitrocellulose membrane in accordance with the procedure used for staphylococcal conjugation, pRW002 was passed to the recipient by mixed-culture transduction. Transfer was inhibited by citrate and serotype B phage antisera but not by DNase I. Cell-to-cell contact was not required, and transfer frequencies increased more than 10-fold in the presence of small concentrations of mitomycin C. These results are consistent with pRW002 transfer in mixed cultures by transduction and not by conjugation or transformation. Immunodiffusion and DNA analyses after agarose gel electrophoresis demonstrated that transductants were exfoliative toxin B producers and housed pRW002. Since mixed-culture transfer has been reported to occur on skin, our results suggest that mixed-culture transduction might be a mechanism for the transfer of genetic determinants for pathogenicity in vivo.

Stimulation of mouse lymphocytes by a mitogen derived from Mycoplasma arthritidis. V. A small basic protein from culture supernatants is a potent T cell mitogen.

Previous studies established that Mycoplasma arthritidis produces a soluble T cell mitogen (MAM), and that response of murine T cells to MAM is genetically restricted. MAM appeared predominantly in the supernatants of senescent cultures, but was not extracted in significant amounts from whole cells. A quantitative assay of MAM activity was devised. MAM formed noncovalent complexes with nucleic acids and uncharacterized high m.w. constituents of sera and of complex media. Partially purified MAM was adsorbed or denatured by glass and plastic surfaces. MAM was protease-labile, had pI greater than or equal to 9, and had Mr ca 15,000 according to gel filtration experiments. MAM was a very minor component of culture supernatant proteins, and even after 200- to estimated 5 X 10(4)-fold purification was not identified as a stainable or ultraviolet-absorbing entity in electrophoretigrams or chromatograms. It was estimated that MAM was half-optimally active at less than 1000th the half-optimal concentration of concanavalin A or phytohemagglutinin. Culture supernatants and highly purified MAM exhibited the same haplotype specificity (H-2k-dependent response) for stimulated proliferation of lymphocytes and for induction of interferon in vitro.

Mutagenesis of extrachromosomal genetic determinants for exfoliative toxin B and bacteriocin R1 synthesis in Staphylococcus aureus after plasmid transfer by protoplast fusion.

In previous studies, we have shown that a 27-megadalton plasmid (pRW002) in Staphylococcus aureus contains genetic determinants for exfoliative toxin B (ET B) and bacteriocin (Bac R1) synthesis and Bac R1 resistance. Attempts to transform or transduce this plasmid to S. aureus or Bacillus subtilis recipients were not successful. However, genetic transfer of the plasmid was possible after polyethylene glycol-induced fusion of S. aureus protoplasts containing pRW002 and S. aureus protoplasts lacking this plasmid. Some of the resulting fusants lost the ability to make ET B, Bac R1, or both products. Fusants that were Bac R1-, Bac R1s, ET B- all lacked the 27-megadalton pRW002 plasmid. The largest class of fusants was Bac R1+, Bac R1r, ET B-. Immunodiffusion analyses of ET B extracts from 28 fusants showed that four ET B+ strains were cross-reacting mutants that produced ET B protein that was serologically related to, but not identical to, the wild-type toxin. Results indicated that genetic transfer of pRW002 after protoplast fusion induced molecular rearrangements that resulted in mutation of the genetic determinants for ET B and Bac R1 synthesis. Recombination of chromosomal genes was enhanced after CaCl2 was added to the protoplast-fusion mixture.

Molecular and serological differentiation of staphylococcal exfoliative toxin synthesized under chromosomal and plasmid control.

Evidence for the existence of two molecular species of exfoliative toxin (ET) synthesized by phage group II Staphylococcus aureus under chromosomal and plasmid control is presented. Serological evidence that these molecular species of toxin are distinct from each other is given. The plasmid-controlled toxin was synthesized along with the chromosomally controlled toxin by the group II UT0002 strain, whereas another group II strain, UT0007, synthesized only the plasmid-controlled toxin. The molecular weight of the plasmid-controlled toxin was slightly less than that of the chromosomally controlled type and could be separated from the latter on 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide slab gels. On 7.5% SDS-polyacrylamide cylindrical gels there was no hint of heterogeneity, and both ETs migrated together as a single homogeneous band. The existence of two serotypes of ET among phage group II strains complicates interpretation of previous work in this field and makes necessary the preparation of two different antigens for radioimmunobinding assays. Discovery of these ET serotypes provided an explanation for previously reported low binding by rabbit hyperimmune serum (B. Wiley, L. Glasgow, and M. Rogolsky, Infect. Immun. 13:513-520, 1976) in the radioimmunobinding test. A molecular species of ET differing from each of the other two serotypes was isolated from cultures of a phage group III S. aureus. This ET produced scalding in suckling mice and was lower in molecular weight than the ET produced under plasmid control by group II strains. Preliminary serological studies indicated that the ET in the group III strain is closely related to or possibly identical to the group II toxin produced under plasmid control.

Production and properties of a staphylococcin genetically controlled by the staphylococcal plasmid for exfoliative toxin synthesis.

Previous data from this laboratory showed that certain phage group 2 staphylococci contain a large 56S virulence plasmid containing genes that code for both exfoliative toxin (ET) and a specific staphylococcin. Optimal cultural conditions for bacteriocin production were similar to those found for ET production. The bacteriocin is an extracellular product produced in small quantities that can be neither extracted from cell pellets with 1 M NaCl nor induced with mitomycin C. The staphylococcin is active against a wide variety of gram-positive organisms and also against group 2 staphylococcal strains that have been cured of the plasmid carrying the staphylococcin marker. The bacteriocin is not inactivated by oxidation, mechanical agitation, or boiling for 15 min. It is sensitive to the action of trypsin and Pronase but not lysostaphin and is stable within a pH range of 4 to 9. It has an isoelectric point of approximately 7.7. Removal of the ampholytes and glycerol from electrofocused staphylococcin preparations resulted in total loss of bacteriocin activity.

Staphylococcal scalded-skin syndrome: development of a primary binding assay for human antibody to the exfoliative toxin.

Exfoliative toxin (ET) from a phage group II Staphylococcus aureus strain causing staphylococcal scalded-skin syndrome was purified by electrofocusing. Ampholytes and salts were removed from the final product by column chromatography on G-50 Sephadex. Sodium dodecyl sulfate-polyacrylamide gels of the final product yielded a single band upon gel electrophoresis, even when 60 mug of protein was placed in the gels. Radiolabeling of the purified toxin with 125I yielded a product that still caused exfoliation of suckling mice, indicating that the toxin was still biologically active. A radioimmunobinding assay was developed and used to test rabbit and human sera for antibodies to exfoliative toxin. Although the maximum percentage of binding was not as high as expected (approximately 40%), it was postulated that either iodination had not been sufficiently vigorous or the toxin had sustained immunological damage. The assay was reproducible and more sensitive than the existing neutralization method and readily applicable to the testing of human sera for exfoliative toxin antibodies.

Phage group II staphylococcal strains with chromosomal and extrachromosomal genes for exfoliative toxin production.

Staphylococcal phage group 2 strain UT0007 was previously shown to contain a high-molecular-weight plasmid containing genes for exfoliative toxin (ET) and bacteriocin production. Phage group 2 strains UT0002 and UT0003 (Tox+Bac-) underwent a twofold and ninefold loss of ET activity, respectively, after growth at 44 C for 18 h. Strain UT0002 also lost total bacteriocin activity. Both strains contained (i) a 56S plasmid that was lost from those substrains showing reduced ET activity and (ii) a 21S plasmid with a gene for cadmium resistance that could be transduced into two recipient strains. Since the ET plasmid-negative substrains still made ET, it was postulated that this residual toxin was made from chromosomal genes. In characterizing the plasmid species from strains UT0002 and UT0003, the 21S but little or no 56S plasmid deoxyribonucleic acid could be isolated after centrifugation of cleared lysates from these strains on dye-buoyant density gradients. Treatment of cleared lysates from strain UT0002 with ethidium bromide, Pronase, or sodium dodecyl sulfate, but not heat at 60 C, induced conversion of the 56S closed circular ET plasmid to a 38S open circular form as determined after centrifugation on 5 to 20% neutral sucrose gradients.

Chromosomal synthesis of staphylococcal exfoliative toxin.

Tox-+ staphylococcal strains, as opposed to Tox-minus strains, produce epidermal exfoliation within 18 h after direct subcutaneous or intraperitoneal injection into newborn mice. The extracellular product responsible for exfoliation is termed exfoliative toxin (ET). When culture supernatant fluid from the plasmid-cured Tox-minus substrains UT 0100 or UT 0111 or from six naturally occurring phage group 2 Tox-minus strains was concentrated 20-fold and inoculated into newborn mice, ET activity could be detected. The Tox-minus, cured derivatives produced ET at levels which were 32 minus and 64-fold lower than the amounts made by their Tox-+ parent strains. Since these Tox-minus, cured substrains contained no plasmid deoxyribonucleic acid, it was postulated that the product possessing ET activity in strains UT 0100 and UT 0111 was made by chromosomal genes. This product has been isolated and purified from strain UT 0100 and appears as two faint bands after electrophoresis on polyacrylamide gels and corresponds in position to a heavy band of ET isolated from the Tox-+ strain UT 0007.

Isolation of extrachromosomal deoxyribonucleic acid for exfoliative toxin production from phage group II Staphylococcus aureus.

The ability of phage group II staphylococcal strain UT 0101 to produce exfoliative toxin and bacteriocin could be eliminated at a high frequency after growth at high temperatures or in the presence of ethidium bromide or sodium dodecyl sulfate. Extrachromosomal deoxyribonucleic acid, associated with the genes for exfoliative toxin and bacteriocin production, was isolated from strain UT 0101 but was absent from an ethidium bromide-cured substrain. The molecular weight of the exfoliative toxin plasmid, determined by co-sedimentation with the penicillinase plasmid, PI258, was 3.3 times 10-7. The 56S covalently closed circular form of the exfoliative toxin plasmid converted to a 38S open circular form after storage or exposure to sodium dodecyl sulfate. Plasmid deoxyribonucleic acid associated with penicillin resistance could not be identified in the penicillin-resistance Tox+ strains, UT 0007 and UT 0001.

Interaction of staphylococcal exfoliative toxin with concanavalin A.

Treatment of mouse embryo fibroblasts (MEF) grown in vitro with purified staphylococcal exfoliative toxin (ET) increased the concanavalin A (Con A) agglutinability of MEF 3.5-fold over control cells. Possible explanations for this phenomenon were investigated. ET lacked proteolytic activity on denatured casein. Con A, however, was found to interact directly with ET as evidenced by the formation of precipitation in an agar gel diffusion plate and in increased turbidity in solution. This interaction was inhibited by alpha-methyl-d-glucopyranoside. The ability of Con A to precipitate with ET suggests that the toxin contains a carbohydrate component and that the carbohydrate associated with ET is branched rather than linear. An analysis of a purified preparation of ET indicated the presence of 9% carbohydrate and no lipid.

Effect of ethidium bromide on elimination of exfoliative toxin and bacteriocin production in Staphylococcus aureus.

The scalded skin syndrome has been associated with phage group II staphylococci. The clinical manifestations of scalded skin syndrome, Ritter's disease, scarlatiniform erythema, and localized bullous impetigo, are due to the production of an extracellular protein, designated exfoliative toxin. Phage group II staphylococci can also produce an extracellular protein, bacteriocin, which is bacteriocidal for specific gram-positive microorganisms. Strain UT 0007 produces both bacteriocin and exfoliative toxin which appear to be products of extrachromosomal genes. These genes are jointly eliminated from strain UT 0007 after growth in either ethidium bromide or at high temperatures.

Staphylococcal scalded skin syndrome: potentiation by immunosuppression in mice; toxin-mediated exfoliation in a healthy adult.

Staphylococcal scalded skin syndrome, associated with exfoliative toxin produced by phage group II Staphylococcus aureus, has recently been reported in an adult receiving immunosuppressive therapy. To determine the effect of immunosuppression on the development of the staphylococcal scalded skin syndrome, experimental animals were treated with prednisolone, azathioprine, or a combination of both drugs utilizing the clinical isolate from the adult with scalded skin syndrome. The mean lethal dose and mean exfoliating dose were identical and were 6,000-fold lower in animals receiving both drugs or azathioprine alone. The isolate was not more virulent and did not produce more toxin than other group II phage-type strains. Furthermore, immunosuppressive therapy failed to enhance the susceptibility of experimental animals to a purified preparation of toxin. Finally, purified exfoliative toxin was demonstrated to produce erythema, Nikolsky's sign, bullous formation, and flaking desquamation in a normal human adult. The results demonstrated the enhanced susceptibility of experimental animals receiving immunosuppressive therapy to the development of the staphylococcal scalded skin syndrome. They further showed that human adults are susceptible to the action of exfoliative toxin and suggested that, in the host with compromised defense mechanisms, toxin-producing strains may invade and initiate infection resulting in toxin production and exfoliation.

Nature of the genetic determinant controlling exfoliative toxin production in Staphylococcus aureus.

Phage group II Staphylococcus aureus has been identified as the etiological agent of the staphylococcal scaleded skin syndrome. The development of an animal model system permitted fulfillment of Koch's postulates and recognition of exfoliative toxin (ET) as being responsible for some of the clinical manifestations of this syndrome. Initial studies directed toward associating a lysogenic phage with the genetic control of ET synthesis failed to support this hypothesis. Growth of two Tox(+) strains at 44 C was more effective than growth in ethidium bromide or sodium dodecyl sulfate in eliminating the ability to produce ET. The early and rapid accumulation of ET-negative (Tox(-)) variants during growth of strain UT 0007 at 44 C, the lack of any selective advantage of the Tox(-) variants over Tox(+) cells during growth at 44 C, and an enhanced elimination frequency at 44 C of 97.9% over the spontaneous frequency of loss strongly suggest that the gene for ET synthesis is extrachromosomal. Additional evidence suggests that this gene is located on a plasmid which is not associated with genes for penicillinase synthesis and cadmium resistance. Two Tox(+) strains harbored lysogenic phage capable of transducing cadmium resistance, but not penicillin resistance, to specific Tox(-) recipients.

Mycoplasma-virus interrelationships in mouse tracheal organ cultures.

The effects produced by single and mixed infections with Mycoplasma pulmonis and influenza A/PR-8 virus were studied in mouse tracheal organ cultures. M. pulmonis multiplied in the tracheal organ cultures, producing inhibition of ciliary activity and histologic tissue damage. The organism grew in close association with the cell membranes but did not appear to attach directly to the membranes or the cilia. Influenza A virus also replicated in tracheal organ cultures, producing ciliary inhibition and more extensive cytopathologic changes. Virus particles were seen by electron microscopy to attach to and cause clumping of the cilia. Simultaneous infection of the organ cultures with mycoplasma and virus resulted in more rapid inactivation of ciliary activity and greater tissue damage than occurred when the cultures were infected with only mycoplasma or virus. Presence of the virus appeared to have no effect on the growth of the mycoplasma; however, the mycoplasma partially interfered with virus replication.

Role of Biological Mimicry in the Pathogenesis of Rat Arthritis Induced by Mycoplasma arthritidis.

Complement fixation (CF), immunofluorescence, and agar gel double-diffusion tests were used to demonstrate an antigenic relationship between rat tissues and Mycoplasma arthritidis. Rabbit antisera against six strains of M. arthritidis exhibited positive reactions in the CF test with an ethyl alcohol-saline extract of rat muscle, whereas only 6 of 18 antisera against other Mycoplasma species were positive. With the use of gel diffusion techniques, absorption of various M. arthritidis antigens with antiserum against rat muscle removed at least one precipitin band when the absorbed mycoplasma antigens were reacted against homologous antisera. Rabbit antiserum against M. arthritidis was conjugated with fluorescein isothiocyanate and reacted against frozen sections of muscle tissues of various animals. As controls, unlabeled normal rabbit serum and rabbit anti-M. arthritidis serum were included to determine the specificity of the reaction. Rat, hamster, and mouse skeletal muscle exhibited specific fluorescence, whereas chicken, beef, frog, and turtle muscles exhibited no specific fluorescence. Mice injected at birth with rat lymphocytes were found to be more susceptible to subsequent infection by M. arthritidis than were normal mice or mice injected at birth with mouse lymphocytes. These results indicate the occurrence of a heterogenetic antigen(s) common to M. arthritidis and rat tissues. Preliminary evidence suggests that this heterogenetic antigen(s) may enable the mycoplasmas to become established in their host.

Immunological response of rodents to murine mycoplasmas.

Mycoplasma arthritidis, M. pulmonis, M. neurolyticum, and M. felis strains administered on various immunization schedules induced complement-fixing and agglutinating antibodies in rats, mice, and guinea pigs. Metabolic inhibiting (MI) antibodies against M. felis were produced by guinea pigs, mice, and rats. M. neurolyticum did not induce MI antibody in mice but induced high titers in guinea pigs and rats. M. pulmonis failed to induce MI antibody titers in mice, induced low titers in rats, and induced high titers in guinea pigs. M. arthritidis induced no or very low titers in rats and low titers in mice, but induced high MI titers in guinea pigs. A positive correlation was demonstrated between ability of the mycoplasma to induce disease and lack of MI antibody formation by its natural host. It is suggested that the apparent inability of the various animals to produce MI antibodies against their natural mycoplasmal parasites may enable the mycoplasmas to become established in their hosts. The concept of biological mimicry is brought forward to explain these observations.

Effect of Animal Passage on Arthritogenic and Biological Properties of Mycoplasma arthritidis.

Determinations of the ED(50) of cultures of Mycoplasma arthritidis disclosed the existence of great diversity in the arthritogenic properties of the various strains. In most cases, the ED(50) values were lowered after passaging in rats, and a more severe arthritis with a shorter period of onset was observed. Heavy suspensions of arthritogenic M. arthritidis did not appear to induce any toxic symptoms. Prior injections of endotoxin did not enhance the toxicity of M. arthritidis suspensions. The more arthritogenic strains grew more slowly in the basal medium and remained viable for longer periods of time than those with lower arthritogenic properties. All strains were identical on the basis of complement-fixation tests. Minor differences observed between strains during gel-diffusion studies could not be correlated with arthritogenic properties. Arthritogenic strains appeared to be less susceptible to the inhibiting action of rabbit antisera than were the nonarthritogenic strains.